Anti-hsv gb monoclonal antibody or antigen-binding fragment thereof

A technology of monoclonal antibodies and binding fragments, applied in the direction of antibodies, antiviral agents, hybrid immunoglobulins, etc., can solve the problems of non-existence of vaccines, increase of drug-resistant virus strains, withdrawal of drugs, etc., to prevent the activation of HSV infection, The effect of treating HSV infection and inhibiting the spread of infection between cells

Pending Publication Date: 2020-05-12
KM生物医薬股份公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is desired to develop a prophylactic vaccine against HSV infection itself, or a therapeutic vaccine to reduce and alleviate infection symptoms and activation symptoms, but there is no effective vaccine at present, and the demand for it is high
[0010] In addition, there is concern that the high-frequency or long-term use of antiviral drugs such as acyclovir and foscarnet sodium will increase drug-resistant virus strains (Non-Patent Document 19)
In addition, these antiviral drugs have various side effects such as abnormal liver function, decreased sperm production ability, digestive tract disorders, and abnormal kidney function, and occasionally, unavoidable dose reduction and drug withdrawal may occur

Method used

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  • Anti-hsv gb monoclonal antibody or antigen-binding fragment thereof
  • Anti-hsv gb monoclonal antibody or antigen-binding fragment thereof
  • Anti-hsv gb monoclonal antibody or antigen-binding fragment thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] The making of embodiment 1 anti-HSV gB monoclonal antibody D48

[0106] Production of scFv-hFc type, Fab type, human-mouse chimeric IgG2a type, and human-guinea pig chimeric IgG2κ for antibody D48 obtained by exhaustive search of anti-HSV-2gB (HSV gB2) antibodies using a human antibody library Antibody D48 of these four molecular forms was further analyzed.

[0107]

[0108] The isolated antibody D48 scFv (single chain Fv, single chain antibody) gene encoding the amino acid sequence shown in SEQ ID NO: 9 was linked to the Fc gene (CH2-CH3) from human IgG1, and cloned into pCAG vector to construct scFv- Fc expression plasmid. For expression, FreeStyle293 or Expi293 expression system (Life Technology Co., Ltd.) was used. The expression plasmid was transfected into the cells, and the culture supernatant was collected after 4-6 days. The culture supernatant was purified with Ab-Rapid PuRe 10 (ProteNova) or Ab-Rapid PuRe Ex (ProteNova) to obtain scFv-hFc.

[0109]

...

Embodiment 2

[0115] Embodiment 2 utilizes the reactivity analysis of the anti-HSV gB antibody of ELISA

[0116]

[0117] The binding activity of the obtained Fab was evaluated by ELISA. Fab was diluted to 2 μg / mL with PBS, 100 μL was added to MaxiSorp plate (Nunc), and incubated at room temperature for 2 hours to immobilize Fab. After immobilization, wash the plate with PBS, serially dilute from 1 μg / mL to 0.316 ng / mL by 3.16 times, add 100 μL to the wells of the recombinant HSV-1gB (gB1-705-strep) plate, and incubate at 37°C. After washing with PBST for 1 hour, 100 µL of detection antibody anti-strep-Tactin (strep-Tactin) / HRP (Funakoshi) was added to the wells of the plate, and incubated at 37°C. After washing with PBST for 1 hour, 100 µL of TMB (3,3',5,5'-tetramethylbenzidine) was added to the wells of the plate to develop color. After 30 minutes, the reaction was terminated with 1N sulfuric acid, and the color development value (O.D.450nm / 650nm) was measured with a microplate reader...

Embodiment 3

[0122] Embodiment 3 utilizes the reactivity analysis of the anti-HSV gB antibody of immunoblotting

[0123] 2 μg / lane of gB1-705-strep was injected into an 8-16% by weight gel for SDS-PAGE, and electrophoresis was performed. After electrophoresis, the gel was transferred to a nitrocellulose membrane (Immobilon-P, MILLIPORE), and blocked with 2% skim milk (Wako)-PBST. After the blocked nitrocellulose membrane was washed with PBST, react with 2% skim milk-PBST, and 10 μg / mL scFv-hFc, Fab, human-mouse chimeric IgG2a or human-guinea pig chimeric IgG2κ at room temperature for 60 minute. After washing again, the nitrocellulose membrane was mixed with anti-hIgG (H+L) / HRP (BIORAD), anti-His tag / HRP (R&D), anti-mouse IgG (H+L) in 2% skim milk-PBST, respectively. / HRP or anti-guinea pig (H+L) / HRP (Invitrogen) reaction, developed with Immobilon Western Detection Regent (Millipore). Produce undenatured gB1-705-strep, denatured gB1-705-strep, and reduced / denatured gB1-705-strep. Reduct...

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Abstract

An anti-HSV monoclonal antibody or an antigen-binding fragment thereof, which is an anti-HSV gB monoclonal antibody specifically binding to the envelope glycoprotein B (gB) of herpes simplex virus (HSV) or an antigen-binding fragment thereof, contains a heavy chain variable region comprising heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 3, heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO: 4 and heavy chain CDR3 having the amino acid sequence represented by SEQ ID NO: 5 and a light chain variable region comprising light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 6, light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 7 and light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 8.

Description

technical field [0001] The present invention relates to an anti-HSV gB monoclonal antibody or an antigen-binding fragment thereof. Background technique [0002] Herpes simplex virus (HSV) is a neurotropic pathogen that transfers to the sensory nerve after first infecting the mucosal epithelium, and latently infects in the trigeminal ganglion or sacral ganglion for life. Latent HSV sometimes activates to cause various diseases (Non-Patent Document 1). [0003] It is known that HSV has two serotypes (HSV-1 and HSV-2). HSV-1 is mainly the cause of lip and corneal herpes, and HSV-2 is mainly the cause of genital herpes. However, in recent years, HSV-1 has frequently become the cause of genital herpes, and HSV-2 has frequently become the cause of cold sores due to diversification of sexual behaviors and the like. In Japan, the antibody-positive (infected) ratio is 60-80% for HSV-1 and 10% for HSV-2. Even if it is limited to HSV-2, the potential demand for the vaccine is estimat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/395A61P31/22C07K16/08C07K16/46C12N1/15C12N1/19C12N1/21C12N5/10C12P21/08
CPCA61P31/22C12N5/10C12N15/09C07K2317/76C07K2317/92C07K16/087C07K2317/21C07K2317/622C07K2317/24C07K2317/55A61K2039/505C07K2317/34C07K16/461C07K2317/51C07K2317/515C07K2317/565
Inventor 森泰亮西村知裕清水裕之河边昭博
Owner KM生物医薬股份公司
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